1. Field of the Invention
The present invention relates to detecting emissions in eluate from a chromatography column, and in particular, to devices for incorporating test samples directly into the flow of the eluate.
2. Description of Related Art
Known emissions detectors can detect activity in the eluate from a chromatography column. These detectors allow eluate to flow through a sample cell located between a pair of photomultipliers. The eluate produces scintillations due to the interaction of radioactive components and solid scintillator located in the sample cell, or because a liquid scintillator is mixed with the eluate upstream of the sample cell. Counts thus detected can be analyzed by a computer-based system that can graphically present the counts as a function of time or as a function of energy. These known analyzers can perform various calculations with the data, and can correct the data based on various criteria.
Injector valves are commonly used in high-performance liquid chromatography (HPLC) as well as many GC systems and other analytical instruments. In HPLC, the injector valves are placed ahead of the chromatography column and serve as the means for placing a measured amount of sample into the column at a precise time. Such valves typically have a single or dual external loops. Loop volumes from perhaps 5 to 100 xcexcL are common, but larger ones can be had. For smaller volumes a groove of suitable size is machined into the valve rotor and there is no external loop.
Multiport valves with or without sample loops have other uses. They have been used as switching valves to interchange chromatography columns while using a single pumping system. They have been used to direct the eluate exiting a column from one detector to another. They have been used to load two columns simultaneously, and to backflush one column while a second is actively used. They have been used to switch from one mobile phase to another.
Conventionally, means are provided to fill a loop while still maintaining mobile phase flow through the column. At the start of a run, a rotor in the valve is moved, which changes the porting so that mobile phase flow is now directed through the loop thereby washing its contents into the column. The valves are made to have minimum dead volumes so that sample is not lost in them and is expressed into the HPLC column without being spread out. Typically they withstand pressures of several thousand psi.
In the past, to check the efficiency of the radioactive detection process, the typical user collects eluate in a vial from a chromatography column at a peak by watching for the peak on a monitor screen, which is providing measurement data from a flow-through detector, a continuous detector of radioactivity connected to the output of the column. The user then takes this vial to a liquid scintillation sample counter and measures it. The peak may or may not be well-defined depending upon the chromatography. Collecting a sample in this fashion will only produce small volumes: just the peak volume of the mobile phase if using solid scintillator, or the mobile phase plus 3-4 additional volumes with liquid scintillator. Most static sample counters require much larger volumes, so the user must dilute the sample with scintillator solution thereby changing the performance from what it was in the flow-through detector. Then, since the user is counting an unknown, that sample is calibrated by addition of an internal standard of known activity and recounting. The overall result is not very accurate. Finally the calibration must be brought back to the original system and entered into the associated software.
Double isotope counting is avoided by many who do not appreciate the mathematics of correcting for spectral overlap in HPLC. One simplification many people make is to spend a great deal of time adjusting their counting windows so that they only look at that portion of the more energetic isotope that lies completely above the most energetic events of the less energetic isotope. Doing that can lose significant counting efficiency, e.g. for 3H/14C dual-label counting, to eliminate the last 1% of 3H in the 14C channel might result in a 10% reduction in 14C counts. If users had a simple way to obtain measurements of the spectral distribution of the different isotopes, they might be more inclined to use such measurement data to calculate corrections for such overlap or spillover.
Variable quench correction is not widely practiced, and then only with liquid scintillator, not with solid (though there is some belief that it should be). First, the sample concentration in an HPLC eluate stream is extremely low; quenching, if any, primarily comes from the composition of the mobile phase itself and variable quenching occurs because the mobile phase is deliberately changed during a run to push different compounds off the column. The normal terminology is xe2x80x9cgradient elutionxe2x80x9d and the gradients often are of different salts and buffers, or water and miscible organic solvents.
Quench correction is not normally practiced for several reasons. Users hope that they will not need the correction, and, because the method seems complex, they do not try to learn whether they really do need this correction. Further, the correction when practiced in the conventional manner, may offer the promise of accuracy, but will require that substantial isotope be consumed, which also discourages frequent repetition.
The method commonly recommended by manufacturers of flow-through detectorsxe2x80x94which is only applicable to liquid scintillation countingxe2x80x94is to make a dummy run with no sample, but otherwise identical in every aspect to the anticipated sample runs. Radioactive standard is added to the scintillator solution prior to the dummy run. The run is made and the activity is counted throughout. Since activity level of the scintillator is known, as well as its flow rate, one can create a tablexe2x80x94from data collected minute by minutexe2x80x94of performance vs. time. When the sample runs are later made, measurements are corrected minute-by-minute using the above performance table.
The problem with this technique is that very large quantities of standard are required. If the chromatography run lasts one-hour with scintillator flowing at 3 ml/min through a 1 ml cell, and one needs to count at 10,000 counts per minute to obtain good statistics and the counting efficiency is about 50% (if it was much higher no one would worry about quenching), the total activity needed is about 3,600,000 disintegrations per minute. That activity is fairly high for a calibrated standard; it does not pre-suppose that this would be done often, yet it is necessary after any change in the analytical procedure: run length, gradient composition, gradient change rate, counting windows, counting cell size, etc.
Percent recovery is also of interest to many users (that is, determining how much of the activity that went into the HPLC column was actually measured in the flow-through detector.) Sometimes activity stays on the HPLC resin, and sometimes it sticks to the stainless steel or plastic tubing. A convenient measurement technique does not currently exist. The operator might pipette a known volume of the original sample mixture into a liquid scintillation vial and count it in a static sample counter. Then the operator must apply all the corrections mentioned above to translate that result to what the flow-through detector gives. It would be better if the raw sample prior to chromatographic separation could be counted in the flow-through detector and each peak subsequently separated is reported as a percent of that sample.
In FIG. 1 of U.S. Pat. No. 4,775,476 eluent passes through 10-port valve 20 to pickup sample through tubular membrane 35. Thereafter, the sample and eluent are directed by the valve through sample loop 27 before arriving at detector 34. In FIG. 2 valve 20 is switched so that eluent is pumped through sample loop 27 to the chromatography column 30, whose eluate is supplied to the detector 34. In this system a sample is not loaded downstream of the chromatography column. Instead, the column is either supplied from the sample loop, or is bypassed while the detector is fed through the sample loop.
In U.S. Pat. No. 4,271,697 sample valve 24 can supply a sample through valve 33 to chromatography column 46, whose eluate is delivered through valve 33 to detector 51. Valve 33 can be switched to backflush column 46 into sample loop 92, associated with valve 34. Loop 92 can supply sample to a second chromatography column 99, whose eluate can be delivered through valve 33 to detector 51. This sample loop is actually between two columns and is therefore unable to directly supply a detector.
U.S. Pat. Nos. 4,840,730; 4,913,821; 4,699,718; and 5,462,660 show sample loops, but these loops are positioned upstream of a chromatography column, in the conventional manner. U.S. Pat. Nos. 5,139,681 and 5,234,599 do not disclose a sample loop, except for an irrelevant mention of a sample loop at column 5, line 63. See also U.S. Pat. No. 4,446,105 (no sample loop).
Accordingly, there is a need for an effective system and method that will allow these, and other useful tests to be outlined herein, to be performed conveniently, so operators will be encouraged to perform them more regularly.
In accordance with the illustrative embodiments demonstrating features and advantages of the present invention, there is provided a chromatography detection system for handling the flow from a chromatography column. The chromatography detection system has an injection valve and a detector for detecting emissions from a flowing sample. The injection valve has at least one loop and is adapted to connect to the chromatography column. The valve is operable to (a) load the at least one loop independently of the chromatography column in a first mode, and (b) serially connect the at least one loop between the chromatography column and the detector in a second mode.
According to another aspect of the invention a method is provided, employing at least one loop and an emissions detector, for handling the flow from a chromatography column. The method includes the step of loading the at least one loop independently of the chromatography column in a first mode. The method also includes the step of serially connecting the at least one loop between the chromatography column and the detector in a second mode. Thus, flow from the at least one loop to the detector is driven by eluate from the chromatography column.
By employing systems and methods of the foregoing type, various tests can be conveniently performed. In one preferred embodiment, a ten port injection valve has one port connected to the outlet of a chromatography column, and another port connected to the inlet of an emissions detector. In some embodiments, the emissions detector can have a T-fitting to introduce a scintillator solution before sending the sample to a sample chamber in a radiochromatography detector, which employs a pair of photomultiplier tubes. Alternatively, the detector can have a solid scintillator in the sample chamber, thereby eliminating the scintillator solution and T-fitting.
Two pairs of ports on the injection valve can connect to two separate loops. Four other ports can be used to fill and drain the two loops. The system is arranged so that with the valve in a first position, one loop is serially connected between the chromatography column and the detector, while the other loop can be filled independently of the chromatography column. When the valve is in a second position, the function of the two loops is essentially interchanged. This arrangement allows an operator to fill each loop with the same or with different solutions. By carefully loading these two loops and by switching them into play at the appropriate time, a variety of tests can be conveniently performed.
An injection from a loop may be done for a sample that might be measured in entirety without chromatographic separation. Alternatively, a standardized quantity of radioactive isotope can be injected directly into the detector for various purposes such as: (1) providing a scale for subsequent measurements from the chromatography column; (2) sending a standard through the detector to either precisely measure efficiency or simply to observe any general trends indicating a change in efficiency; (3) more sophisticated tests that sequentially inject different isotopes or different eluates to enable correction for more subtle phenomenon (spillover and quenching).
For isocratic operation or for gradient runs where the efficiency does not change significantly (which is most often the case), two loops enable determination of single- or dual-isotope efficiency, as well as spillover for dual-isotope counting. For quench correction, dual isotope correction could be facilitated, although this is an infrequent requirement.
In a simple embodiment, the user will manually fill the loop and then control the injection with a handle or a pushbutton. Other embodiments can automate the filling and can rely on programmed actuation.
By using an injector valve with separate loops, one can conveniently determine efficiency by loading into a loop, the same internal standard as would be used to check a vial of eluate at a separate liquid scintillation sample counter, as described previously. The result should be equivalent to that obtained when measuring an unknown. Here however, the counting can be advantageously performed with the same instrument, with the mobile phase being the same, and with the scintillator (solid or liquid) being the same. Then, once the process is over, the result can be manually or automatically incorporated into any software used for correction of that run and all subsequent runs, until a new standard injection is made.
Systems of the foregoing type can be used to evaluate the effect of spillover in dual-isotope chromatography. For example, chromatography can be performed with samples containing a mixture of isotopes, often 3H and 14C. By using the injection valve with one loop filled with a 3H standard and the other filled with a 14C standard one can be much less exacting with the window settings for the energy spectrum. (Actually, in a practical plumbing arrangement, where the mobile phase must continue to flow before the actual run starts, the second loop will start as a bypass and will typically be filled after the first one is drained.) When the first loop is drained, the operator can determine how much 3H falls in each channel. Then, when the second loop is drained, the operator can determine how much 14C is in each channel. With that information, preferably a computer will perform the proper mathematics and automatically incorporate the results into subsequent runs.
Also, with arrangements of the foregoing type, one can evaluate efficiency in spite of gradient elution. It is possible to make periodic injections during a dummy run, record the performance, and then use interpolation between the points. A few intermittent injections may not be quite as comprehensive as making measurements across the entire run, but such thoroughness may be unnecessary. One would expect the most popular usage to be xe2x80x9cbefore and afterxe2x80x9d injections to verify that there has not been significant quenching. Even such sparse measurements are an improvement over the conventional situation, where checks are difficult and extremely rare, and the unverified assumption is made that nothing has changed during the run. Also, using the above injection valve will allow checking the performance of packed cells during gradient elution, something not being done by any means at this time.
An operator can also determine percent recovery with the preferred injection valve. When a run starts, before the first peak appears (almost always there is a delay of several minutes) the combined sample can be pushed out of the loop through the detector and the total count recorded. It is then a simple matter to report each peak as a percent of that total. Because a typical loop may be 20 xcexcL while the amount of sample in the run is 50 xcexcL, or some other amount, one must make provision for a normalization factor, but essentially that is all.
In practice, the preferred injection valve provides a quick response. Typically, within a few seconds of the valve actuation, there are sharp, well-defined peaks. If any of these standards/samples entered the detector via an HPLC column instead of from the downstream loop, there would be a delay of minutes to sometimes an hour or more until a peak appeared. Moreover, chromatography tends to spread peaks and flatten them or sometimes make them overlap, which is why one would not want to involve the HPLC system in any of these measurements, even if it were possible.
In these situations (efficiency, quench correction, etc.) the preferred plumbing arrangement allows one or both loops to always be connected to a reservoir of standard activity. If only one isotope is used, one loop merely serves as a bypass. If both single-isotope efficiency calibration, and single-isotope quench correction are practiced, the same standard solution will serve for both. The quantity of standard solution required is remarkably little. With 10 xcexcL loops, and with wastage being four times the loop volume, 10 mL of standard solution will enable 200 calibrations (which may be more than are now performed over the life of many instruments).
It is considered simplest and best that both efficiency and quench calibrations be performed in dummy runs which precede runs of unknown samples. Efficiency runs need only last for short periods, a few minutes at most, but quench calibration runs must mimic the anticipated sample runs as to time and gradient.